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Myocyte enhancer factor (MEF)-2 plays essential roles in T-cell transformation associated with HTLV-1 infection by stabilizing complex between Tax and CREB.

Jain P, Lavorgna A, Sehgal M, Gao L, Ginwala R, Sagar D, Harhaj EW, Khan ZK - Retrovirology (2015)

Bottom Line: Herein, utilizing virus-infected primary CD4+ T cells and the virus-producing cell line, MT-2, we describe the involvement and regulation of Myocyte enhancer factor-2 (specifically MEF-2A) during the course of HTLV-1 infection and associated disease syndrome.MEF-2 stabilization of Tax/CREB complex was confirmed by a novel promoter-binding assay that highlighted the involvement of NFAT (nuclear factor of activated T cells) in this process via Tax-mediated activation of calcineurin (a calcium-dependent serine-threonine phosphatase).MEF-2-integrated signaling pathways (PI3K/Akt, NF-κB, MAPK, JAK/STAT, and TGF-β) were also activated during HTLV-1 infection of primary CD4+ T cells, possibly regulating MEF-2 activity.

View Article: PubMed Central - PubMed

ABSTRACT

Background: The exact molecular mechanisms regarding HTLV-1 Tax-mediated viral gene expression and CD4 T-cell transformation have yet to be fully delineated. Herein, utilizing virus-infected primary CD4+ T cells and the virus-producing cell line, MT-2, we describe the involvement and regulation of Myocyte enhancer factor-2 (specifically MEF-2A) during the course of HTLV-1 infection and associated disease syndrome.

Results: Inhibition of MEF-2 expression by shRNA and its activity by HDAC9 led to reduced viral replication and T-cell transformation in correlation with a heightened expression of MEF-2 in ATL patients. Mechanistically, MEF-2 was recruited to the viral promoter (LTR, long terminal repeat) in the context of chromatin, and constituted Tax/CREB transcriptional complex via direct binding to the HTLV-1 LTR. Furthermore, an increase in MEF-2 expression was observed upon infection in an extent similar to CREB (known Tax-interacting transcription factor), and HATs (p300, CBP, and p/CAF). Confocal imaging confirmed MEF-2 co-localization with Tax and these proteins were also shown to interact by co-immunoprecipitation. MEF-2 stabilization of Tax/CREB complex was confirmed by a novel promoter-binding assay that highlighted the involvement of NFAT (nuclear factor of activated T cells) in this process via Tax-mediated activation of calcineurin (a calcium-dependent serine-threonine phosphatase). MEF-2-integrated signaling pathways (PI3K/Akt, NF-κB, MAPK, JAK/STAT, and TGF-β) were also activated during HTLV-1 infection of primary CD4+ T cells, possibly regulating MEF-2 activity.

Conclusions: We demonstrate the involvement of MEF-2 in Tax-mediated LTR activation, viral replication, and T-cell transformation in correlation with its heightened expression in ATL patients through direct binding to DNA within the HTLV-1 LTR.

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Related in: MedlinePlus

MEF-2 co-localizes with Tax in the nucleus of HTLV-1 transformed cell lines. MT-2 cells (A) and C8166 cells (B) were cultured overnight on poly-L-lysine coated glass coverslips. The cells were then fixed, permeabilized and then stained with anti-Tax, anti-MEF-2, anti-CREB or anti-IRAK1 as indicated. Nuclei were stained with DAPI and cells were subjected to confocal microscopy.
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Fig5: MEF-2 co-localizes with Tax in the nucleus of HTLV-1 transformed cell lines. MT-2 cells (A) and C8166 cells (B) were cultured overnight on poly-L-lysine coated glass coverslips. The cells were then fixed, permeabilized and then stained with anti-Tax, anti-MEF-2, anti-CREB or anti-IRAK1 as indicated. Nuclei were stained with DAPI and cells were subjected to confocal microscopy.

Mentions: To confirm that MEF-2 interacts with Tax not only in solution but also within the infected cells, we performed confocal microscopy using two HTLV-1 transformed cell lines, MT-2 (Figure 5A) and C8166 (Figure 5B). Tax localized in the cytoplasm and nucleus in both cell lines. pCREB as a positive control strongly co-localized with Tax in the nucleus. Tax and MEF-2A also co-localized within the nuclear compartment. As a negative control, a cytoplasmic protein (IRAK1) without any known interaction with Tax was used. As expected, this protein did not co-localize with Tax in either cell type.Figure 5


Myocyte enhancer factor (MEF)-2 plays essential roles in T-cell transformation associated with HTLV-1 infection by stabilizing complex between Tax and CREB.

Jain P, Lavorgna A, Sehgal M, Gao L, Ginwala R, Sagar D, Harhaj EW, Khan ZK - Retrovirology (2015)

MEF-2 co-localizes with Tax in the nucleus of HTLV-1 transformed cell lines. MT-2 cells (A) and C8166 cells (B) were cultured overnight on poly-L-lysine coated glass coverslips. The cells were then fixed, permeabilized and then stained with anti-Tax, anti-MEF-2, anti-CREB or anti-IRAK1 as indicated. Nuclei were stained with DAPI and cells were subjected to confocal microscopy.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4374383&req=5

Fig5: MEF-2 co-localizes with Tax in the nucleus of HTLV-1 transformed cell lines. MT-2 cells (A) and C8166 cells (B) were cultured overnight on poly-L-lysine coated glass coverslips. The cells were then fixed, permeabilized and then stained with anti-Tax, anti-MEF-2, anti-CREB or anti-IRAK1 as indicated. Nuclei were stained with DAPI and cells were subjected to confocal microscopy.
Mentions: To confirm that MEF-2 interacts with Tax not only in solution but also within the infected cells, we performed confocal microscopy using two HTLV-1 transformed cell lines, MT-2 (Figure 5A) and C8166 (Figure 5B). Tax localized in the cytoplasm and nucleus in both cell lines. pCREB as a positive control strongly co-localized with Tax in the nucleus. Tax and MEF-2A also co-localized within the nuclear compartment. As a negative control, a cytoplasmic protein (IRAK1) without any known interaction with Tax was used. As expected, this protein did not co-localize with Tax in either cell type.Figure 5

Bottom Line: Herein, utilizing virus-infected primary CD4+ T cells and the virus-producing cell line, MT-2, we describe the involvement and regulation of Myocyte enhancer factor-2 (specifically MEF-2A) during the course of HTLV-1 infection and associated disease syndrome.MEF-2 stabilization of Tax/CREB complex was confirmed by a novel promoter-binding assay that highlighted the involvement of NFAT (nuclear factor of activated T cells) in this process via Tax-mediated activation of calcineurin (a calcium-dependent serine-threonine phosphatase).MEF-2-integrated signaling pathways (PI3K/Akt, NF-κB, MAPK, JAK/STAT, and TGF-β) were also activated during HTLV-1 infection of primary CD4+ T cells, possibly regulating MEF-2 activity.

View Article: PubMed Central - PubMed

ABSTRACT

Background: The exact molecular mechanisms regarding HTLV-1 Tax-mediated viral gene expression and CD4 T-cell transformation have yet to be fully delineated. Herein, utilizing virus-infected primary CD4+ T cells and the virus-producing cell line, MT-2, we describe the involvement and regulation of Myocyte enhancer factor-2 (specifically MEF-2A) during the course of HTLV-1 infection and associated disease syndrome.

Results: Inhibition of MEF-2 expression by shRNA and its activity by HDAC9 led to reduced viral replication and T-cell transformation in correlation with a heightened expression of MEF-2 in ATL patients. Mechanistically, MEF-2 was recruited to the viral promoter (LTR, long terminal repeat) in the context of chromatin, and constituted Tax/CREB transcriptional complex via direct binding to the HTLV-1 LTR. Furthermore, an increase in MEF-2 expression was observed upon infection in an extent similar to CREB (known Tax-interacting transcription factor), and HATs (p300, CBP, and p/CAF). Confocal imaging confirmed MEF-2 co-localization with Tax and these proteins were also shown to interact by co-immunoprecipitation. MEF-2 stabilization of Tax/CREB complex was confirmed by a novel promoter-binding assay that highlighted the involvement of NFAT (nuclear factor of activated T cells) in this process via Tax-mediated activation of calcineurin (a calcium-dependent serine-threonine phosphatase). MEF-2-integrated signaling pathways (PI3K/Akt, NF-κB, MAPK, JAK/STAT, and TGF-β) were also activated during HTLV-1 infection of primary CD4+ T cells, possibly regulating MEF-2 activity.

Conclusions: We demonstrate the involvement of MEF-2 in Tax-mediated LTR activation, viral replication, and T-cell transformation in correlation with its heightened expression in ATL patients through direct binding to DNA within the HTLV-1 LTR.

Show MeSH
Related in: MedlinePlus